Subsets of interferon signaling and response to immune checkpoint blockade.
Authors: Daniel Lee, Brooke Horowitch, Min Ding, Sandra Martinez-Morilla, Thazin Nwe Aung, Feriel Ouerghi, Xueting Wang, Wei Wei, William Damsky, Mario Sznol, Harriet M. Kluger, David L. Rimm, Jeffrey Joseph Ishizuka
Journal: Journal of Clinical Oncology (2023)

9522 Background: Interferon (IFN) signaling in tumors is critical for both response and resistance to immune checkpoint inhibitors (ICI). While PD-L1 is an approved biomarker for ICI response in some cancers, most have no approved biomarkers for ICI response. Further, there are no clinical strategies to predict IFN sensitive vs resistant states. We hypothesized that distinct patterns of IFN signaling in melanoma, including STAT1 (precursor to IFN signaling), phosphorylated STAT1 (pSTAT1, marker of active IFN signaling), and PD-L1 (canonical inhibitory output), are associated with response or resistance to ICI. Methods: Samples from 2 tissue microarrays of 97 patients total with metastatic melanoma who received ICI from 2011-2017 were randomized into discovery [D] or validation [V] cohorts. Multiplexed immunofluorescent microscopy was used to assess STAT1, pSTAT1, and PD-L1. 54 tumors were assessed for CD3. Signals were quantified via AQUA (Automated Quantitative Analysis). High vs low signals were defined around the discovery cohort median AQUA scores. Treatment response was assessed using RECIST, and 3-year survival was analyzed. For in vitro studies, 4 human melanoma cell lines were stimulated with IFNγ/β, and Western blots were performed for STAT1, pSTAT1, and PD-L1. Results: Median follow up time was 20.2 mos (range 0.9-95.4 mos). Higher overall histospot, tumor, and nuclear STAT1 were in responders (CR/PR/SD &gt; 6 mos) compared to non-responders (SD &lt; 6 mos/PD, histospot p = 0.029 [D] and 0.040 [V]) and were associated with improved survival (histospot HR 0.25 [95% CI 0.08-0.76] p = 0.015 [D]; HR 0.32 [95% CI 0.12-0.84] p = 0.021 [V]). PD-L1 and pSTAT1 did not validate as associated with ICI response/survival. While many tumors had concordant high/low STAT1, pSTAT1, and PD-L1, many also had a discordant STAT1 or PD-L1 predominance. To assess the basis of this discordance, we performed IFN stimulation and Western blots of melanoma cell lines. IFNβ upregulated STAT1 greater than PD-L1 in both acute and chronic settings (p &lt; 0.001 and &lt; 0.001), while chronic IFNγ upregulated PD-L1 compared to the acute setting (p = 0.039) but not STAT1 (p = 0.105). Therefore, we hypothesized patients with discordant STAT1 and PD-L1 expression may have differential responses to ICI. In the overall cohort, STAT1 high PD-L1 high tumors (IFN engaged) were associated with improved survival compared to STAT1 low PD-L1 low tumors (IFN low, HR 0.28 [95% CI 0.11-0.70] p = 0.007). Interestingly, STAT1 high PD-L1 low tumors were associated with improved survival compared to STAT1 low PD-L1 high tumors (HR 0.28 [95% CI 0.08-0.94] p = 0.04). Accordingly, STAT1 high PD-L1 high and STAT1 high PD-L1 low tumors had greater CD3 AQUA scores compared to STAT1 low PD-L1 low tumors (p &lt; 0.001 and p = 0.018 respectively). Conclusions: Combined STAT1 and PD-L1 expression patterns may better predict melanoma response to ICI and provide insight into IFN sensitive versus resistant states.